UW News

August 15, 2010

Genetic alterations common to Kabuki syndrome discovered through streamlined DNA sequencing

UW Health Sciences/UW Medicine

Using a new, rapid and inexpensive DNA sequencing strategy, scientists have discovered genetic alterations that may account for a majority of Kabuki syndrome cases.

Kabuki syndrome is a rare congenital disorder. Children born with this condition often have distinctive facial features — elongated eyes and arched eyebrows — that resemble actors’ make-up for Kabuki, a Japanese theatrical performance. The syndrome is also characterized by a range of mild to moderate intellectual difficulties and physical problems, such as heart, skeletal and kidney malformations.

The researchers studied genetic samples from 10 unrelated children diagnosed with the syndrome. They started by sequencing just the exome — the protein-coding parts of a gene that make up only about 1 to 2 percent of the human genome — to try to locate genetic alterations that could be associated with Kabuki syndrome.

The findings were published in the Aug. 15 advanced online edition of Nature Genetics. The work was carried out by scientists at the University of Washington in Seattle as part of a larger effort to use second generation DNA sequencing technologies to identify genes for 20 rare, single-gene disorders. The project is funded by a $3.9 million American Recovery and Reinvestment Act grant from the National Human Genome Research Institute, part of the National Institutes of Health.

“It’s clear that second generation DNA sequencing technologies are a powerful and effective tool that scientists can use to accelerate the discovery of genes for rare diseases, an effort that previously was slow and costly,” said Dr. Eric D. Green, director of the National Human Genome Research Institute. “The newfound ability to rapidly find genes that cause more than 6,000 rare diseases is an important step forward for researchers who are trying to understand the biology of these conditions and thereby improve strategies to care for patients they affect.”

The study reported today identified mutations in the MLL2 gene as a cause of Kabuki syndrome.

While the details of how this gene normally works are enigmatic, it appears to be one of the regulators of embryonic development in mammals. MLL2 produces an enzyme that is part of a complex network controlling if and when certain genes will be activated in the cells of the growing embryo.

“The particular type of protein that it turned out to be was surprising,” said senior author Dr. Jay Shendure, assistant professor of genome sciences at the University of Washington (UW) in Seattle. “The findings also suggest that epigenetic programming [events or agents that switch genes on or off or determine their timing] may play a role in this disorder, which is certainly a clue for further investigation into how mutations in this gene change its function.”

Kabuki syndrome has an estimated incidence of 1 in 32,000 individuals, and about 400 cases have been reported worldwide. The syndrome was first described by Japanese scientists in 1981, including geneticist Dr. Norio Niikawa, who is also one of the senior researchers on this week’s report on the MLL2 mutations linked to the disorder. The disease is not exclusive to people of Asian descent; it affects people from other geographical origins as well. In this study, seven of the affected children were of European parentage, two were Hispanic and one of was of mixed European-Haitian ancestry.

“The discovery of the mutations in MLL2 are a major step forward in understanding the origins of Kabuki syndrome and should facilitate diagnostic testing,” said Dr. Michael Bamshad, UW professor of pediatrics in the Division of Genetics and Development and another senior researcher on the study. “But more importantly, it now gives us the opportunity to study a mechanism that underlies Kabuki syndrome to assess potential therapeutic agents.” Bamshad practices at Seattle Children’s Hospital, where he sees patients and families with genetic disorders.

The lead authors of the study published this week are Sarah B. Ng, a UW graduate student in genome sciences, and Abigail W. Bigham, a UW postdoctoral fellow in pediatrics. The research team began with the premise that Kabuki syndrome was caused by alterations in just a single gene. They compared the 10 exomes from the children to the reference human genome sequence to discover instances where all affected individuals shared a gene containing novel variants. The researchers found none.

The results prompted the researchers to assume that variants in any of several genes could be the cause. So, they looked for novel variants in genes shared across subsets of the 10 exomes. For instance, novel variants were shared in three genes in nine patients’ exomes, six genes shared in at least eight exomes, and 16 genes shared among seven exomes.

With no obvious way to rank these gene candidates, the researchers categorized each Kabuki syndrome case based on a subjective assessment of the degree to which the child had the distinctive facial characteristics and whether the patients had developmental delays or physical malformations.

The researchers found mutations that led to a loss of function in the MLL2 gene in the four highest ranked cases, and in three of the remaining six cases. These errors result in the production of shortened, nonfunctional proteins.

Second-generation sequencing technologies have some difficulty identifying some types of genetic variants that lead to a loss of function in genes. Follow-up sequencing using the traditional Sanger DNA sequencing method, capable of reliably detecting loss of function variants, was able to identify additional MLL2 gene variants in two of three remaining cases. Ultimately, the researchers were able to discover MLL2 variations in nine of the 10 exomes.

The findings were validated using Sanger methods to sequence the protein-coding portions of the MLL2 gene for 43 additional Kabuki syndrome cases. Novel variants in MLL2 were found in 26 of the 43 cases. In the end, a total of 33 distinct mutations in the MLL2 gene were found in 35 of 53, or 66 percent, of families with Kabuki syndrome.

“This disease was notably harder to tackle than the last one,” Shendure said, in reference to the discovery reported in November of the mutations behind Miller syndrome, the first time the cause of a rare, single-gene disorder was located using exome sequencing. “As a consequence, the process of figuring out Kabuki syndrome mutations helped us to further develop the techniques and analytical tools used to interpret exome sequencing data.”

The researchers also learned that in each of 12 cases of Kabuki syndrome for which DNA from both parents was available, the MLL2 variations were newly mutated in the affected child’s genome and not present in the parents’ genomes.

“Our findings suggest that alterations in the MLL2 gene are a major cause of Kabuki syndrome,” said Shendure. “Furthermore, it is clear that there may be additional genes that cause Kabuki syndrome. To discover them, it will be important to sequence the exomes of additional well-characterized cases of Kabuki syndrome in which MLL2 mutations are not present.”

In addition to funding from the National Human Genome Research Institute, individual researchers on the team were supported by grants from the National Heart Lung and Blood Institute, the National Institute of Environmental Health Sciences and the Eunice Kennedy Shriver National Institute of Child Health and Human Development. The researchers thank the patients and their families and the Kabuki Syndrome Network for their participation.

Along with Ng, Bigham, Nickerson, Shendure, and Bamshad, other researchers on the study are Kati J Buckingham, Mark C Hannibal, Margaret McMillin, Heidi Gildersleeve, Anita E Beck, Holly K Tabor, and Heather C Mefford, all of the UW Department of Pediatrics; Greg M Cooper, Choli Lee, Emily H Turner, Josh D Smith, and Mark J Rieder, all of the UW Department of Genome Sciences; Koh-ichiro Yoshiura, Nagasaki University, Japan; Naomichi Matsumoto, Yokohama City University, Japan; and Tohru Ohta and Norio Niikawa, both of the Health Sciences University of Hokkaido, Japan. Hannibal, Beck and Tabor are also affiliated with Seattle Children’s Research Institute.



To learn more about exome sequencing and its applications to disease research, view a video, http://www.nhlbi.nih.gov/recovery/researchers/index.php?id=200, of one of the Kabuki syndrome paper’s authors, Dr. Deborah Nickerson, professor of genome sciences and director of the Northwest Genomics Center, University of Washington. Nickerson has received Recovery Act funds from the National Heart, Lung, and Blood Institute (NHLBI) to perform exome sequencing on DNA samples from NHLBI-funded population studies of common heart, lung, and blood diseases.



The public may request useful information about genetic and rare diseases by contacting the Genetic and Rare Diseases Information Center (GARD), http://www.genome.gov/10000409, which provides immediate access to experienced information specialists who can furnish current and accurate information in both English and Spanish.





NHGRI is one of the 27 institutes and centers at the NIH, an agency of the Department of Health and Human Services. The NHGRI Division of Intramural Research develops and implements technology to understand, diagnose and treat genomic and genetic diseases. Additional information about NHGRI can be found at its Web site, www.genome.gov.


The National Institutes of Health (NIH) — The Nation’s Medical Research Agency — is the primary federal agency for conducting and supporting basic, clinical and translational medical research, and it investigates the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit www.nih.gov.